LIGHT, a member of the TNF superfamily, induces morphological changes and delays proliferation in the human rhabdomyosarcoma cell line RD.

LIGHT is a member of the tumor necrosis factor (TNF) superfamily, which binds two known receptors, lymphotoxin-beta receptor (LTbetaR) and the herpesvirus entry mediator (HVEM)/TR2. We investigated the effects of LIGHT on the human rhabdmyosarcoma cell line RD. LIGHT delayed cell proliferation and induced morphological changes of the cells. These effects were not shown by other TNF family ligands such as TNFalpha and LTalpha, which induced the transcriptional activity of nuclear factor-kappaB (NF-kappaB) and NF-kappaB-responsible chemokine productions in the same manner as did LIGHT. LTalpha1beta2, another TNF family ligand for LTbetaR, was shown to have similar activities in RD cells as LIGHT. Both LIGHT and LTalpha1beta2 induced the expression of muscle-specific genes such as smooth muscle (SM) alpha-actin, while TNFalpha and LTalpha did not. These findings indicate that LIGHT may be a novel inducer of RD cell differentiation associated with SM alpha-actin expression through the LTbetaR.

Evaluation of posttreatment response of hepatocellular carcinoma with contrast-enhanced coded phase-inversion harmonic US: comparison with dynamic CT.

PURPOSE: To assess the reliability of contrast material-enhanced real-time gray-scale ultrasonography (US) in evaluating posttreatment response of hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Fifty HCC nodules were examined with contrast-enhanced coded phase-inversion harmonic US before and after treatment. Intratumoral vascularity was assessed with continuous imaging in the early arterial phase and with interval-delay scanning to depict tumor parenchymal flow during the blood pool phase. Vascular findings at US were compared with those at dynamic computed tomography (CT). RESULTS: In 50 HCC nodules before treatment, positive enhancement of tumor vessels and tumor parenchymal flow (stain) were observed in 47 (94%) and 46 (92%), respectively. Either tumor vessel or stain was visualized with coded harmonic US in 49 of 50 nodules. Eighty-one coded harmonic US studies were performed in 49 posttreatment HCC nodules. Compared with dynamic CT, the sensitivity, specificity, and accuracy of coded harmonic US in helping to detect positive enhancement in pretreatment HCC were 98% (49 of 50), 100% (50 of 50), and 98% (49 of 50), respectively. After treatment, positive enhancement of tumor vascularity was observed in 39 (48%) of 81 posttreatment studies, and no enhancement was observed in others (52%). Coded harmonic US demonstrated partial and no enhancement of tumor vascularity in four and one nodule, respectively; after transcatheter arterial embolization with iodized oil, evaluation of tumor vascularity with dynamic CT was difficult because of the presence of oil. CONCLUSION: With enhancement, coded harmonic US depicted tumor vascularity by showing tumor vessels in a real-time fashion at continuous imaging and tumor parenchymal flow at interval-delay scanning.

Sonographic diagnosis of pancreatic islet cell tumor: value of intermittent harmonic imaging.

We describe a case of nonfunctioning islet cell tumor of the pancreas diagnosed preoperatively by intermittent harmonic power Doppler imaging and digital subtraction gray-scale harmonic imaging and the use of the contrast agent SH U 508A (Levovist). Hypervascularity and tumor perfusion were clearly demonstrated with both harmonic imaging techniques in the early arterial phase. Sonographic findings were confirmed by other modalities and by histopathologic examination.

Metastin suppresses the motility and growth of CHO cells transfected with its receptor.

We recently reported having identified of the ligand for an orphan G-protein-coupled receptor, hOT7T175, as the gene product (68-121)-amide of the metastasis suppressor gene KiSS-1. We further showed that the ligand, which we named "metastin," inhibits chemotaxis and invasion of Chinese hamster ovary (CHO) cells transfected with hOT7T175 cDNA (CHO/h175) in vitro, and pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. In the present study, we investigated the activity of metastin in CHO/h175 cells in greater detail. Metastin significantly suppressed motility in a chemotaxis assay and wound healing assay at 10-100 nM order concentrations. Two N-terminally truncated peptides, metastin(40-54) and metastin(45-54) inhibited the migration of CHO/h175 cells as potently as metastin itself. Metastin also inhibited the spreading, monolayer growth and colony formation in agar (0.8%) of CHO/h175 cells at 10-100 nM concentrations. These results indicate that metastin is a potent inhibitor of cell motility, leading to suppression of cell growth and antimetastatic activity, and suggest that low molecular chemical compounds could replace its activity as a novel antimetastatic agent.

The generation and characterization of a cell line derived from a sporadic renal angiomyolipoma: use of telomerase to obtain stable populations of cells from benign neoplasms.

Angiomyolipomas are benign tumors of the kidney derived from putative perivascular epithelioid cells, that may undergo differentiation into cells with features of melanocytes, smooth muscle, and fat. To gain further insight into angiomyolipomas, we have generated the first human angiomyolipoma cell line by sequential introduction of SV40 large T antigen and human telomerase into human angiomyolipoma cells. These cells show phenotypic characteristics of angiomyolipomas, namely differentiation markers of smooth muscle (smooth muscle actin), adipose tissue (peroxisome proliferator-activator receptor gamma, PPARgamma), and melanocytes (microophthalmia, MITF), thus demonstrating that a single cell type can exhibit all of these phenotypes. These cells should serve as a valuable tool to elucidate signal transduction pathways underlying renal angiomyolipomas.

Hepatocellular carcinoma: depiction of tumor parenchymal flow with intermittent harmonic power Doppler US during the early arterial phase in dual-display mode.

PURPOSE: To assess the effectiveness of contrast material-enhanced intermittent harmonic Doppler ultrasonography (US) in depicting tumor vessels and tumor parenchymal flow (stain) in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Fifty-eight patients with 65 HCC nodules were examined by using intermittent harmonic power Doppler US and digital subtraction harmonic B-mode US, both with intravenous administration of SH U 508A. Vascular findings at early arterial phase harmonic US were classified as positive enhancement or nonenhancement, depending on the tumor vascularity relative to the surrounding liver parenchyma. These results were compared with those of three-phase helical dynamic computed tomography (CT). RESULTS: For hypervascular HCCs, there was excellent depiction of tumor vessels and tumor stain with the two intermittent harmonic US methods. The sensitivity and specificity for depiction of tumor vascularity were 93% (41 of 44 nodules) and 100% (21 of 21), respectively, with intermittent harmonic power Doppler US and 86% (38 of 44) and 100% (21 of 21), respectively, with subtraction US, as compared with these values at dynamic CT. Attenuation was an important factor in the depictability of tumor vascularity at harmonic US. CONCLUSION: Contrast-enhanced intermittent harmonic US enables noninvasive demonstration of tumor vessels and especially tumor stain in HCC.

Possible risk reduction in esophageal cancer associated with MPO -463 A allele.

Myeloperoxidase (MPO), an enzyme found in lysosomes of phagocytes, causes hydroxy radicals linked to DNA damage and activation of smoking related carcinogens. A -463 G/A polymorphism in the promoter region of the MPO gene results in reduced gene expression, which would imply lower susceptibility of esophageal cancer in mutant carriers. We conducted case-control study to test this hypothesis. Cases were 91 patients with esophageal cancer and controls were 241 non-cancer outpatients. MPO genotypes were examined by PCR-RFLP. The allele frequency for MPO -463A was found to be 8.2% for cases and 10.5% for controls. The age, sex, smoking and drinking status adjusted odds ratio for all subjects for MPO -463 GG/GA as compared to the AA was 0.61 (95% CI: 0.28-1.32). The adjusted odds ratio for the GG/GA genotype was significantly low (0.15; 0.03-0.76, P=0.022) for those aged 61 years or older who had a significantly higher odds ratio for smoking than younger subjects. No difference was observed in disease risk when prevalent and incident cases were compared. Although there are limitations for interpretation of this study because of prevalent case-control study and partial statistical significance, these results suggest that MPO -463 A allele reduce the risk of esophageal cancer.

A novel secreted tumor antigen with a glycosylphosphatidylinositol-anchored structure ubiquitously expressed in human cancers.

In a search for novel genes expressed in human cancers, we identified one gene from an assembled expressed sequence tag database. Northern blot analysis revealed that the gene, termed alcan, was expressed in various types of human cancer cell lines and in the fetus, but not in normal tissues. The alcan gene is located on chromosome 6 and is encoded on a 246-amino-acid protein with weak homology to classical major histocompatibility complex class I. Its gene product, ALCAN, had hydrophobic amino acid clusters at both the N- and C-terminal regions and was predicted to be a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Flow cytometric analysis revealed that ALCAN was detected on the surface of human cancer cells and on alcan-transfected CHO-K1 cells. ALCAN was also secreted from these cells, suggesting that some portion of the molecules was secreted by enzymatic cleavage by, for example, phospholipases. Mutational analysis of ALCAN suggested that the GPI-anchored position was the Ser(216) residue. These findings indicate that ALCAN may be a potential target for cancer diagnosis or therapy.

Stimulation effect of galanin-like peptide (GALP) on luteinizing hormone-releasing hormone-mediated luteinizing hormone (LH) secretion in male rats.

Galanin-like peptide (GALP) is a recently isolated hypothalamic peptide which has sequence homology to galanin and binds to galanin receptors with high affinity. It has been shown that GALP neurons are localized in the arcuate nucleus and that GALP-immunoreactive fibers are in close apposition with LHRH neurons in the medial preoptic area (MPA). In the present study, we found that intracerebroventricular (icv) administration of GALP increased the plasma LH level but did not change the levels of other hormones. Concomitantly, accumulation of c-Fos protein was dramatically increased in the nuclei of LHRH-positive cells in the MPA by icv GALP administration. Furthermore, the GALP-induced plasma LH response was completely abolished by pretreatment with Cetrorelix, a LHRH receptor antagonist. On the other hand, GALP did not affect the release of LH, FSH, TSH, ACTH, GH or PRL directly from dispersed rat pituitary cells in vitro. These results strongly suggest a role for GALP in the control of gonadotropin secretion through a hypothalamic mechanism involving the release of LHRH.

Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor.

Metastasis is a major cause of death in cancer patients and involves a multistep process including detachment of cancer cells from a primary cancer, invasion of surrounding tissue, spread through circulation, re-invasion and proliferation in distant organs. KiSS-1 is a human metastasis suppressor gene, that suppresses metastases of human melanomas and breast carcinomas without affecting tumorigenicity. However, its gene product and functional mechanisms have not been elucidated. Here we show that KiSS-1 (refs 1, 4) encodes a carboxy-terminally amidated peptide with 54 amino-acid residues, which we have isolated from human placenta as the endogenous ligand of an orphan G-protein-coupled receptor (hOT7T175) and have named 'metastin'. Metastin inhibits chemotaxis and invasion of hOT7T175-transfected CHO cells in vitro and attenuates pulmonary metastasis of hOT7T175-transfected B16-BL6 melanomas in vivo. The results suggest possible mechanisms of action for KiSS-1 and a potential new therapeutic approach.

Molecular properties of apelin: tissue distribution and receptor binding.

We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [

Conformation of a peptide ligand bound to its G-protein coupled receptor.

Many peptide hormones elicit a wide array of physiological effects by binding to G-protein coupled receptors. We have determined the conformation of pituitary adenylate cyclase activating polypeptide, PACAP(1--21)NH(2), bound to a PACAP-specific receptor by NMR spectroscopy. Residues 3--7 form a unique beta-coil structure that is preceded by an N-terminal extended tail. This beta-coil creates a patch of hydrophobic residues that is important for receptor binding. In contrast, the C-terminal region (residues 8--21) forms an alpha-helix, similar to that in the micelle-bound PACAP. Thus, the conformational difference between PACAP in the receptor-bound and the micelle-bound states is limited to the N-terminal seven residues. This observation is consistent with the two-step ligand transportation model in which PACAP first binds to the membrane nonspecifically and then diffuses two-dimensionally in search of its receptor; a conformational change at the N-terminal region then allows specific interactions between the ligand and the receptor.

Contrast-enhanced subtraction harmonic sonography for evaluating treatment response in patients with hepatocellular carcinoma.

OBJECTIVE: Our objective was to assess the usefulness of contrast-enhanced subtraction harmonic sonography in evaluating the treatment response of patients with hepatocellular carcinoma. SUBJECTS AND METHODS: Thirty-two hepatocellular carcinoma lesions in 26 patients (age range, 44-85 years; mean age, 66 years) were examined with Levovist-enhanced intermittent harmonic imaging before and after therapy. A Toshiba Powervision 8000 was used. A subtraction image was obtained by digitally subtracting the last-frame harmonic image from the first-frame image when multishot mode was preset. Results of contrast-enhanced CT were compared with the results of subtraction harmonic imaging. RESULTS: Before therapy, an enhancement pattern of tumor vascularity was seen for 93.8% (30/32) of hepatocellular carcinoma nodules on subtraction harmonic imaging. After therapy, subtraction harmonic imaging showed 46.7% (14/30) enhancement (incomplete tumor necrosis) and 53.3% (16/30) no enhancement (complete tumor necrosis). When dynamic CT was the gold standard, the sensitivity, specificity, and accuracy of subtraction harmonic imaging were 93.3%, 100%, and 96.7%, respectively. Intratumoral flow signals in hepatocellular carcinoma after therapy on harmonic imaging were used as a guide to target additional percutaneous therapy. CONCLUSION: Digital subtraction contrast-enhanced harmonic imaging can depict tumor vascularity in hepatocellular carcinoma after therapy sensitively and accurately. Because it is easy to perform and provides real-time needle insertion guidance, it may be preferable to perform after localized therapy to monitor treatment response, which will reduce unnecessary CT scanning.

Quantitative analysis of gene expressions related to inflammation in canine spastic artery after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The possible role of inflammatory reaction of the cerebral artery in the pathogenesis of cerebral vasospasm has been noted in recent studies. We quantitatively measured the levels of expression of genes related to inflammation in the spastic artery in a canine double-hemorrhage model. METHODS: Twenty dogs were assigned to 4 groups: group D0, control; group D2, dogs killed 2 days after cisternal injection of blood; group D7, dogs given double cisternal injections of blood and killed 7 days after the first injection; and group D14. Angiography was performed twice: on the first day and before the animals were killed. Total RNA was extracted from the basilar artery. The expressions of interleukin (IL)-1alpha, IL-6, IL-8, IL-10, tumor necrosis factor-alpha, E-secretin, fibronectin, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, transforming growth factor-ss, basic fibroblast growth factor, and collagen types I, III, and IV were examined with TaqMan real-time quantitative reverse transcription-polymerase chain reaction. RESULTS: Prolonged arterial narrowing peaking on 7 day was observed. There was a significant difference in vessel caliber between D0, D2, D7, and D14 groups (P:<0.0001). There were significant differences in mRNA expression in the basilar artery for IL-1alpha, IL-6, IL-8, ICAM-1, and collagen type I between D0, D2, D7, and D14 groups (P:=0.0079, 0. 0196, 0.0040, 0.0017, and <0.0001, respectively). The average level of mRNA was highest in D7 for IL-1alpha, IL-6, IL-8, and ICAM-1 (17-, 16-, 131-, and 1.7-fold compared with those of D0, respectively) and in D14 for collagen type I (10.9-fold). CONCLUSIONS: Increased expression of genes related to inflammation in the spastic artery suggests that inflammatory reaction of the cerebral artery is associated with sustained contraction.

Analyses for susceptibility of rat anterior pituitary cells to prolactin-releasing peptide.

We validated the effect of prolactin-releasing peptide (PrRP) on prolactin (PRL) secretion from rat anterior pituitary cells in in vitro culture. We found that culture conditions considerably influenced the response of the anterior pituitary cells to PrRP. Longer culture term (4 d) was required to obtain better responses of the anterior pituitary cells to PrRP in comparison to thyrotropin-releasing hormone (TRH). Under the culture conditions employed here, PrRP was comparable to TRH in the potency promoting PRL secretion, and the action of PrRP was very specific for PRL secretion. The susceptibility of the anterior pituitary cells to PrRP varied in female rats depending on the process of reproduction: the cells prepared from lactating rats were the most sensitive to PrRP compared with those from random-cycle and pregnant rats. Because the expression levels of PrRP receptor mRNA in the pituitary varied during the reproductive process, we speculated that the susceptibility of the anterior pituitary cells would reflect cellular changes including the expression level of PrRP receptors. In addition, treatment with estrogen in vivo enhanced the susceptibility of the cultured anterior pituitary cells in male rats. Our results indicate that the susceptibility of the rat anterior pituitary cells to PrRP is regulated by physiological mechanisms.

Identification of genes differentially expressed in canine vasospastic cerebral arteries after subarachnoid hemorrhage.

To understand the molecular processes of continuous vasospasm of cerebral arteries after subarachnoid hemorrhage, mRNA differential display and screening of cDNA expression array were performed to identify genes that are differentially expressed in vasospastic arteries of canine two-hemorrhage models. The expression levels of 18 genes were found to be upregulated, and those of two genes to be downregulated. Of these, 12 represent known genes or homologues of genes characterized previously, and the other eight genes are not related to any sequences in the databases. The known genes include five upregulated inflammation-related genes encoding monocyte chemotactic protein-1, cystatin B, inter-alpha-trypsin inhibitor family heavy chain-related protein, serum amyloid A protein, and glycoprotein 130, suggesting that inflammatory reaction may be involved in the development of cerebral vasospasm. The upregulation of three known genes encoding stress-related proteins of vascular endothelial growth factor, BiP protein, and growth-arrest and DNA-damage-inducible protein may be involved in possible cell survival in the damaged arteries. A full-length cDNA for the unknown clone DVS 27, whose expression was most highly upregulated, was isolated from the cerebral artery cDNA library by hybridization. Characterization of these genes should help to clarify the molecular mechanism of continuous cerebral vasospasm after subarachnoid hemorrhage.

Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum.

By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.

Tsc2(+/-) mice develop tumors in multiple sites that express gelsolin and are influenced by genetic background.

Tuberous sclerosis (TSC) is an autosomal dominant genetic disorder in which benign hamartomas develop in multiple organs, caused by mutations in either TSC1 or TSC2. We developed a murine model of Tsc2 disease using a gene targeting approach. Tsc2-null embryos die at embryonic days 9.5-12.5 from hepatic hypoplasia. Tsc2 heterozygotes display 100% incidence of multiple bilateral renal cystadenomas, 50% incidence of liver hemangiomas, and 32% incidence of lung adenomas by 15 months of age. Progression to renal carcinoma, fatal bleeding from the liver hemangiomas, and extremity angiosarcomas all occur at a rate of less than 10%. The renal cystadenomas develop from intercalated cells of the cortical collecting duct and uniformly express gelsolin at high levels, enabling detection of early neoplastic lesions. The tumor expression pattern of the mice is influenced by genetic background, with fewer large renal cystadenomas in the outbred Black Swiss background and more angiosarcomas in 129/SvJae chimeric mice. The slow growth of the tumors in the heterozygote mice matches the limited growth potential of the great majority of TSC hamartomas, and the influence of genetic background on phenotype correlates with the marked variability in expression of TSC seen in patients.

Isolation and identification of melanin-concentrating hormone as the endogenous ligand of the SLC-1 receptor.

Melanin-concentrating hormone (MCH), which is an orexigenic peptide, was isolated and identified as the endogenous ligand of the SLC-1 receptor. We established a CHO cell line expressing the rat SLC-1 receptor to search for its endogenous ligand. The extract of rat whole brain showed inhibition of intracellular forskolin-induced cAMP accumulation in rat SLC-1-expressing CHO cells and was purified. Using HPLC purification, we isolated and identified MCH as the endogenous ligand of the SLC-1 receptor. The authentic MCH demonstrated a dose-dependent inhibitory effect on cAMP accumulation in forskolin-stimulated rat and human SLC-1-expressing CHO cells with an EC(50) value of 0.2 nM for both the rat and human SLC-1 receptors. This is the first description of the functional receptor for MCH.

Clinical outcome in localized renal cell carcinomas related to immunoexpression of proliferating cell nuclear antigen, Ki-67 antigen, and tumor size.

This study was designed to clarify the relationship between clinical outcome and immunoexpression of proliferating cell nuclear antigen (PCNA) and Ki-67 antigen (Ki-67) in 71 localized renal cell carcinomas (RCCs) of Robson stage I and II, related to the disease recurrence and tumor size. PCNA and Ki-67 expressions showed significant differences between non-recurring and recurring groups and more variability in stage II than in stage I. Recurrence rates according to tumor size were 0% for /=5.0 cm. It was concluded that PCNA and Ki-67 expression profiles were considered to be closely related to tumor stage and showed some promise for predicting the disease recurrence.